Yasutaka Hayashida

FUNCTIONAL BIOENGINEERED CORNEAL EPITHELIAL SHEET GRAFTS FROM CORNEAL STEM CELLS EXPANDED EX VIVO ON A TEMPERATURE - RESPONSIVE CELL CULTURE SURFACE

Background. Limbal stem-cell deficiency by ocular trauma or diseases causes corneal opacification and visual loss. Recent attempts have been made to fabricate corneal epithelial graft constructs, but the technology is still evolving. We have developed a novel cell-sheet manipulation technology using temperature-responsive culture surfaces to generate functional, cultivated corneal epithelial cell sheet grafts. Methods. Human or rabbit limbal stem cells were cocultured with mitomycin C -treated 3T3 feeder layers on temperature-responsive culture dishes at 37°C. Cell sheets were harvested from the dishes after 2 weeks by reducing temperature to 20°C. Histologic analyses, immunoblotting, and colony-forming assay were performed to characterize the cell sheets. Autologous transplantation was undertaken to reconstruct the corneal surfaces of rabbits with experimentally induced limbal stem cell deficiencies. Results. Multilayered corneal epithelial sheets were harvested intact simply by reducing the temperature, without the use of proteases. Cell-cell junctions and extracellular matrix on the basal side of the sheet, critical to sheet integrity and function, remained intact. A viable population of corneal progenitor cells, close in number to that originally seeded, was found in the sheets. Harvested sheets were easily manipulated, transplantable without any carriers, and readily adhesive to corneal stroma so that suturing was not required. Corneal surface reconstruction in rabbits was highly successful. Conclusions. Cell sheet engineering technology allows us to create intact, transplantable corneal epithelial cell sheets that retain stem cells from limbal stem cells expanded ex vivo. Our research indicates highly promising clinical capabilities for our bioengineered corneal epithelial sheet.

Functional human corneal endothelial cell sheets harvested from temperature-responsive culture surfaces

This study reports a new method for fabricating bioengineered human corneal endothelial cell sheets suitable for ocular surgery and repair. We have initially cultured human corneal endothelial cells on type IV collagen‐coated dishes and, after several passages, expanded cells were then seeded onto novel temperature‐responsive culture dishes. Four weeks after reaching confluence, these cultured endothelial cells were harvested as intact monolayer cell sheets by simple temperature reduction without enzymatic treatment. Scanning electron microscopy indicated that these cells were primarily hexagonal with numerous microvilli and cilia, similar to the native corneal endothelium. The Na+, K+‐ATPase pump sites were located at the cell borders as in vivo. Moreover, cell densities and numbers of pump sites were identical to those of in vivo human corneal endothelium under optimized conditions. A 3H‐ouabain binding analysis demonstrated a linear proportionality for cell pump density between confluent cell densities of 575 cells/mm2 and 3070 cells/mm2. We also confirmed Na+, K+‐ATPase activity in the sheets in vitro. Xenograft transplantation results showed that the fabricated sheets retain their function of maintaining proper stromal hydration in vivo. We have established a regimen to culture and proliferate human corneal endothelial cells and fabricate endothelial sheets ex vivo morphologically and functionally similar to the native corneal endothelium. Our results support the value of harvested cell sheets for clinical applications in ocular reconstructive surgery in patients with ocular endothelial decompensation.

Human Amniotic Epithelial Cells as Novel Feeder Layers for Promoting Ex Vivo Expansion of Limbal Epithelial Progenitor Cells

Human amniotic epithelial cells (HAECs) are a unique embryonic cell source that potentially can be used as feeder layers for expanding different types of stem cells. In vivo, HAECs uniformly expressed pan‐cytokeratins (pan‐CK) and heterogeneously expressed vimentin (Vim). The two phenotypes expressing either pan‐CK(+)/Vim(+) or pan‐CK(+)/Vim(‐) were maintained in serum‐free media with high calcium. In contrast, all HAECs became pan‐CK(+)/Vim(+) in serum‐containing media, which also promoted HAEC proliferation for at least eight passages, especially supplemented with epidermal growth factor and insulin. Mitomycin C‐arrested HAEC feeder layers were more effective in promoting clonal growth of human limbal epithelial progenitors than conventional 3T3 murine feeder layers. Cells in HAEC‐supported clones were uniformly smaller, sustained more proliferation, and expressed less CK12 and connexin 43 but higher levels of stem cell‐associated markers such as p63, Musashi‐1, and ATP‐binding cassette subfamily G2 than those of 3T3‐supported clones. Subculturing of clonally expanded limbal progenitors from HAEC feeder layers, but not from 3T3 feeder layers, gave rise to uniformly p63‐positive epithelial progenitor cells as well as nestin‐positive neuronal‐like progenitors. Collectively, these results indicated that HAECs can be used as a human feeder layer equivalent for more effective ex vivo expansion of adult epithelial stem cells from the human limbus.

Matrix morphogenesis in cornea is mediated by the modification of keratan sulfate by GlcNAc 6-O-sulfotransferase

Matrix assembly and homeostasis in collagen-rich tissues are mediated by interactions with proteoglycans (PGs) substituted with sulfated glycosaminoglycans (GAGs). The major GAG in cornea is keratan sulfate (KS), which is N-linked to one of three PG core proteins. To ascertain the importance of the carbohydrate chain sulfation step in KS functionality, we generated a strain of mice with a targeted gene deletion in Chst5, which encodes an N-acetylglucosamine-6-O-sulfotransferase that is integral to the sulfation of KS chains. Corneas of homozygous mutants were significantly thinner than those of WT or heterozygous mice. They lacked high-sulfated KS, but contained the core protein of the major corneal KSPG, lumican. Histochemically stained KSPGs coassociated with fibrillar collagen in WT corneas, but were not identified in the Chst5-null tissue. Conversely, abnormally large chondroitin sulfate/dermatan sulfate PG complexes were abundant throughout the Chst5-deficient cornea, indicating an alteration of controlled PG production in the mutant cornea. The corneal stroma of the Chst5-null mouse exhibited widespread structural alterations in collagen fibrillar architecture, including decreased interfibrillar spacing and a more spatially disorganized collagen array. The enzymatic sulfation of KS GAG chains is thus identified as a key requirement for PG biosynthesis and collagen matrix organization.

Characterization and Comparison of Intercellular Adherent Junctions Expressed by Human Corneal Endothelial Cells in Vivo and in Vitro

PURPOSE Human corneal endothelial cell (HCEC) proliferation is controlled by HCEC junctions, but the mechanism of proliferation remains unknown. The authors sought to characterize adherent junction components of in vivo HCECs and to compare their gene expression and their proliferative potential with those of in vitro counterparts. METHODS Stripped human Descemet membranes were digested with collagenase A, and the resultant HCEC aggregates were cultured for 7, 14, and 21 days in supplemented hormonal epithelial medium (SHEM). The growth of HCEC monolayers was monitored by BrdU labeling performed 24 hours before termination. In vivo and in vitro HCECs were subjected to immunostaining to FITC-phalloidin and antibodies to different junction components and BrdU. Their mRNA expressions were determined by RT-PCR. RESULTS In vivo HCECs expressed transcripts of N-, VE-, E-, and P-cadherins, alpha-, beta-, gamma-, and p120-catenins, and p190. In vitro HCEC counterparts also expressed all these mRNAs except P-cadherin. In vivo HCECs displayed continuous circular F-actin, N-cadherin, beta- and p120-catenins, and p190, discontinuous circular VE-cadherin bands at or close to cell junctions, and E-cadherin in the cytoplasm. Such an in vivo pattern was gradually achieved by in vitro HCECs at day 21 and was correlated with a progressive decline of BrdU labeling. CONCLUSIONS In vivo and in vitro HCECs displayed distinct protein cytolocalization of N-, VE-, and E-cadherins, beta- and p120-catenins, and p190. Progressive maturation of adherent junctions was associated with a decline of the proliferative potential. This information allows us to devise new strategies to engineer in vitro HCECs by targeting these components.

Suppression of Activation and Induction of Apoptosis in RAW264.7 Cells by Amniotic Membrane Extract

PURPOSE Macrophages play a pivotal role in initiating, maintaining, and resolving host inflammatory/immune responses but may cause recalcitrant inflammation and tissue damage if not controlled. Clinically, amniotic membrane (AM) transplantation suppresses inflammation in ocular surface reconstruction. Experimentally, the authors and others have reported that AM facilitates macrophage apoptosis. However, it remains unclear whether such anti-inflammatory activity is retained in AM extract (AME). METHODS Herein the authors demonstrate in resting and activated (by interferon [IFN]-gamma, lipopolysaccharide [LPS], or IFN-gamma/LPS) murine monocyte/macrophage RAW264.7 cells that AME suppresses cell spreading and reduces actin filaments determined by phalloidin staining and Western blotting of Triton X-100 extracted cell lysate. RESULTS Western blot and immunocytochemistry staining showed AME downregulates the expression of such cell surface markers as CD80, CD86, and major histocompatibility complex class 2 antigen. Cell growth/viability is inhibited whereas cell apoptosis is enhanced by AME. Accordingly, secreted proinflammatory cytokines such as TNF-alpha and IL-6 are reduced, but anti-inflammatory cytokine IL-10 is upregulated. CONCLUSIONS Collectively, these results suggest that, similar to amniotic membrane, AME retains anti-inflammatory activities and does so by downregulating activation and inducing apoptosis in macrophages.

Surgical Strategies for Fornix Reconstruction Based on Symblepharon Severity

PURPOSE To identify surgical strategies of fornix reconstruction for symblepharon graded according to the length from the limbus to the lid margin, to the width, and to associated inflammation. DESIGN Retrospective, comparative, interventional case series. METHODS In 61 eyes with symblepharon, cicatrix lysis and amniotic membrane transplantation (AMT) were performed with sutures (n = 34) or fibrin glue (n = 27) together with (n = 47) or without (n = 14) intraoperative mitomycin C (MMC), plus fornix reconstruction using anchoring sutures without (n = 30) or with (n = 7) oral mucosal graft or with conjunctival autograft (n = 4). Overall, success was defined as an outcome of complete success (restoration of an anatomically deep fornix) or partial success (focal recurrence of scar), and failure was defined as the return of symblepharon. RESULTS For a follow-up of 25 +/- 10.8 months, the overall success was achieved by the first attempt in 52 eyes (85.2%) and failure resulted in nine eyes (14.8%); however, the success rate improved to 59 eyes (96.7%) with additional attempts. At the first attempt, AMT alone achieved overall successes in 92.8% of grade I eyes and in 100% of grade II eyes. Additional anchoring sutures achieved successes in 100% of grade I eyes, 70% of grade II eyes, and 71.4% of grade III/IV eyes. Additional oral mucosa or conjunctival autograft achieved successes in 100% of grade III/IV eyes. The complete success was correlated positively with lower grades of symblepharon or intraoperative use of MMC, but negatively correlated with younger ages, canthal involvement, or use of anchoring sutures. Anatomic improvement was accompanied by reduction of preoperative conjunctival inflammation (n = 40), improved visual acuity (n = 14), improved ocular motility (n = 18), improved eyelid closure (n = 3), and feasibility of contact lens wear (n = 10). CONCLUSIONS Successful outcome can be achieved by selectively deploying cicatrix lysis and AMT, intraoperative MMC, anchoring sutures, and oral mucosal or conjunctival autograft based on the severity of pathogenic symblepharon.

The use of amniotic membrane in reducing adhesions after strabismus surgery

Postoperative adhesions after strabismus surgery may influence surgical outcome. Different techniques have been used with varying success to reduce these adhesions. We describe a novel surgical technique in which amniotic membrane is used to wrap the extraocular muscles.

Air Exposure–Induced Squamous Metaplasia of Human Limbal Epithelium

PURPOSE Squamous metaplasia is a pathologic process that frequently occurs in nonkeratinized stratified ocular surface epithelia. The mechanism for this occurrence is largely unknown except for vitamin A deficiency. METHODS Human limbal explants were cultured under airlift with or without p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 or in a submerged manner for different durations up to 2 weeks. Epithelial cell proliferation, differentiation, limbal stem cell maintenance, and expansion were studied using certain markers such as Ki67, p63, K10 and K12 keratins, filaggrin, Pax6, ABCG-2, and Musashi-1. Expression of phospho-p38 MAPK and its downstream transcription factors, C/EBPalpha and C/EBPbeta, were studied by immunohistochemistry. Epithelial cells harvested from explants after 2 weeks of culturing under different conditions were seeded onto 3T3 feeder layers and cultured for 12 days. The differentiation of clonal epithelial cells was investigated by double staining to K12 and K10 keratins. RESULTS The squamous metaplasia model was successfully created by culturing human limbal explants at an air-liquid interface (airlift) for 2 weeks. Increased stratification and hyperproliferation only happened in the limbal, but not the corneal, epithelium in airlift, but not submerged, cultures. Epithelial proliferation was associated with a transient increase of limbal epithelial stem cells. Abnormal epidermal differentiation-evidenced by positive expression of K10 keratin in suprabasal cells and filaggrin in superficial cells-ensued. Clones generated from epithelial cells harvested from airlift culture only expressed K12 keratin without K10. As early as 2 days in airlift cultures, p38 expression emerged in limbal basal epithelial cells and gradually extended to the cytoplasm and nuclei. Furthermore, addition of the p38 inhibitor SB203580 abolished abnormal epidermal differentiation without affecting limbal epithelial proliferation. Expression of C/EBPalpha and C/EBPbeta, downstream of the p38 MAPK signaling pathway, was strongly induced by airlift culture and partially was inhibited by SB203580. CONCLUSIONS Dryness resulting from exposure activates p38 MAPK signaling coupled with abnormal epidermal differentiation without intrinsic alteration of stem cells in the limbus. On the ocular surface, p38 inhibitors may have the potential to revert the pathologic process of squamous metaplasia induced by dryness.

Enzymes Responsible for Synthesis of Corneal Keratan Sulfate Glycosaminoglycans

Keratan sulfate glycosaminoglycans are among the most abundant carbohydrate components of the cornea and are suggested to play an important role in maintaining corneal extracellular matrix structure. Keratan sulfate carbohydrate chains consist of repeating N-acetyllactosamine disaccharides with sulfation on the 6-O positions of N-acetylglucosamine and galactose. Despite its importance for corneal function, the biosynthetic pathway of the carbohydrate chain and particularly the elongation steps are poorly understood. Here we analyzed enzymatic activity of two glycosyltransferases, β1,3-N-acetylglucosaminyltansferase-7 (β3GnT7) and β1,4-galactosyltransferase-4 (β4GalT4), in the production of keratan sulfate carbohydrate in vitro. These glycosyltransferases produced only short, elongated carbohydrates when they were reacted with substrate in the absence of a carbohydrate sulfotransferase; however, they produced extended GlcNAc-sulfated poly-N-acetyllactosamine structures with more than four repeats of the GlcNAc-sulfated N-acetyllactosamine unit in the presence of corneal N-acetylglucosamine 6-O sulfotransferase (CGn6ST). Moreover, we detected production of highly sulfated keratan sulfate by a two-step reaction in vitro with a mixture of β3GnT7/β4GalT4/CGn6ST followed by keratan sulfate galactose 6-O sulfotransferase treatment. We also observed that production of highly sulfated keratan sulfate in cultured human corneal epithelial cells was dramatically reduced when expression of β3GnT7 or β4GalT4 was suppressed by small interfering RNAs, indicating that these glycosyltransferases are responsible for elongation of the keratan sulfate carbohydrate backbone.